Enterotoxigenic (ETEC) and verotoxigenic (VTEC) Escherichia coli (E. coli) are important causes of disease in man and animal.
By adhering to the mucosa, bacteria are prevented from being eradicated by the natural cleaning mechanisms of the host such as intestinal peristaltism and secretion of fluid. Furthermore, by being in close contact with the mucosal surfaces, the bacteria have better access to the available nutrients and the secreted toxins are delivered close to their target tissue. Attachment to host tissues is mediated by adhesins expressed on the surface of the microbial organism. The bacterial adhesins are known to be lectins that combine with complementary carbohydrates on the host cell surfaces. The bacterial adhesins are part of typical polymeric structures that are named fimbriae or pili. Fimbriae are thin, flexible filaments with a diameter of 2 to 4 nm without axial hole, whereas pili are rigid structures with diameter of 7-8 nm with axial hole. Both are composed of major subunits that build up the fimbrial shaft complemented with minor subunits that mediate adhesion or have a structural function. In most cases, the adhesin is a minor subunit.
Typically, adhesins consist of two domains: an N-terminal carbohydrate-specific lectin domain and a C-terminal pilin domain. The crystal structures reveal that the lectin domains of these fimbrial adhesins all have an immunoglobulin-like (Ig-like) fold in common, although they show little to no sequence identity. The C-terminal pilin domain connects the adhesin to the fimbrial shaft and shares the highly conserved and incomplete Ig-like fold of the other fimbrial subunits.
Some of the carbohydrates that are recognized by bacterial adhesins have been successfully determined in the past (e.g. those of type 1 and P pili, F5 fimbriae, F17 fimbriae and S fimbriae), whereas other carbohydrate receptor structures remain unsolved as for F4 fimbriae and F6 fimbriae.
Type 1 pili adhesins demonstrate specificity for mannose, but they have a considerable higher affinity for oligosaccharides such as Manα3Manβ4GlcNAc and Manα6(Manα3)Manα6(Manα3)Man. The biological receptor for the adhesin of type 1 pili (FimH) in the urinary tract is the glycoprotein uroplakin Ia that is strongly expressed by differentiated uroepithelial cells. In addition, the Tamm-Horsfall glycoprotein that is secreted in human urine is recognized by type 1 pili, providing a first line of defense against urinary tract infections.
P fimbriated E. coli causing urinary tract infections have specificity for galabiose (Galα4Gal) present in the globoseries of glycolipids on for instance human erythrocytes and uroepithelial cells. The minimum receptor isotype is called globotriasylceramide (GbO3), which is the galabiose residue linked by a β-glucose residue to a ceramide group that anchors the receptor in the membrane. Other members of the globoseries, namely GbO4 and GbO5, were also found to be recognized by 3 adhesins expressed by PapG alleles (PapGI, PapGII and PapGIII), each adhesin demonstrating a different specificity for a different receptor isotype. Whereas most bacterial lectins recognize terminal non reducing sugars, the P-fimbriated E. coli adhesins also recognize internal sugars.
In addition to P pili, other mannose-resistant adhesins, for instance S fimbriae, have been identified in E. coli strains associated with human urinary tract or septic infection. These fimbriae bind to sialyl(α2,3)galactosides on various glycoproteins of human erythrocytes and kidney epithelium, but not on lipids.
F5 fimbriae adhesins, associated with enterotoxigenic E. coli that causes diarrhea in neonatal pigs, calves and lambs, recognize NeuGcα2-3Galβ4GlcNAc. This glycolipid receptor structure was first isolated from equine erythrocytes, but is also detected in mucosal scrapings of piglet and calf intestine, thus functioning as in vivo receptor for F5+ E. coli. 
Not all carbohydrate receptors of pathogenic E. coli strains have been identified in the past. Studies to point out the carbohydrate binding specificity of F4+ E. coli have not been conclusive so far. The antigenic variants F4ab, F4ac and F4ad each bind their own carbohydrate structure. The sequence Galα3Gal is recognized by F4ab fimbriae, whereas Galβ3GalNAc and Galβ3/4GlcNAc are essential parts of the receptor sites of the F4ac variant.
The receptor molecules for fimbrial lectins are glycoprotein or glycolipid in nature. To investigate the glycoprotein receptors involved in adhesion of various bacteria, gelelectrophoresis and immunoblotting techniques are used, followed by overlay assays. Glycolipid receptors for bacterial adhesion are identified and characterized after separation by thin-layer chromatography.
It is known that the carbohydrates being recognized by type 1 pili and S fimbriae are anchored on proteins, whereas the carbohydrate receptors for P pili and F5 fimbriae are located on lipids. The saccharides of glycolipids are easier to determine since each lipid molecule carries only one saccharide chain, in contrast with glycoproteins that may have several N- or O-linked carbohydrate chains on one molecule.
Expression of receptors for pathogenic bacteria is confined to restricted areas of the body, determining the place where infection may occur. In some cases, receptors are only expressed during a limited period in life.
The sugar specificity of fimbriae sometimes is not solely determined by the adhesin, but can also be modulated by the fimbrial shaft.
Interaction between the adhesin and the receptor can activate signal transduction processes that alter gene expression in both bacterial and host cells.
In newly weaned pigs, F18 fimbriated E. coli producing entero- and/or verotoxins induce diarrhea and/or edema disease, counting for substantial economical losses in pig industry.
F18 fimbrial structures are expressed by F18+ E. coli strains that mediate adhesion with the intestinal brush border. F18 fimbriae are expressed by the fed (fimbriae associated with edema disease) gene cluster and are typically composed of multiple copies of the major subunit FedA whereas a minor subunits FedF is only present in small amounts. FedF was determined to be the adhesive subunit.
The binding domain of FedF is located at the amino-terminal half of the protein, as found in many other bacterial adhesins. The region essential for binding was mapped between amino acid 60 and 109 and the charged amino acid residues Lys-72, His-88 and His-89 were found to be important for receptor binding. These residues could either be directly involved in interaction with the receptor or could influence the tertiary structure of the binding pocket.
A crucial step in pathogenesis of F18+ E. coli is the initial attachment to a specific receptor (F18R) on the porcine intestinal epithelium. Some pigs are found to be resistant to colonization by F18+ E. coli due to lack of F18R expression. The F18R status of pigs is genetically determined with the gene controlling expression of the F18R mapped to the halothane linkage group on pig chromosome 6. This locus contained two candidate genes, namely FUT1 and FUT2, encoding α(1,2)fucosyltransferases and expression analysis of both genes in the porcine small intestine revealed that the FUT2 gene was differentially expressed, while the FUT1 gene was expressed in all examined pigs. Sequencing of the FUT1 gene of pigs being either susceptible or resistant to F18+ E. coli infections showed a polymorphism (G or A) at nucleotide 307. Presence of the A nucleotide on both alleles (FUT1A/A genotype) led to significantly reduced enzyme activity and corresponds with the F18+ E. coli resistant genotype, whereas susceptible pigs had either the heterozygous FUT1G/A or the homozygous FUT1G/G genotype. These findings have led to the development of a PCR-RFLP test to differentiate between F18R positive and F18R negative pigs. This test is described in U.S. Pat. No. 6,596,923 which was published on 22 Jul. 2003.
This DNA-test was shown to correlate well with in vivo susceptibility to F18+ E. coli infections and is therefore considered to be valuable in predicting the F18R phenotype. However, although an attractive approach to eliminate F18+ E. coli infections, genetic selection for F18+ E. coli resistant pigs was not routinely used in Belgian pig farms because of reports of genetic association with the stress susceptibility allele (RYR1T) in the Swiss Landrace. Indeed, the genes encoding the F18R were found to be close to the locus for stress susceptibility on porcine chromosome 6. However, by performing positive selection for F18+ E. coli resistance in Swiss pigs, the frequency of the resistant FUT1A allele could be raised to 0.42, while originally not more than 5 to 10% of this population was resistant.
When investigating the association between FUT1A and RYR1T alleles more thoroughly in the Belgian pig population, it was found that they were not associated, revealing new potential for this prevention strategy.
Although creating an F18+ E. coli resistant pig population could solve the problem of F18+ E. coli infections in pigs in a permanent way, it still needs to be ascertained that no co-selection of unwanted traits, other than stress susceptibility, occurs. In addition, it will take several years, if not decades to establish such a population. So, other receptor-based anti-adhesive therapy against F18+ E. coli infections could be a great benefit for pig industry.
Several other approaches have been undertaken to eliminate F18+ E. coli infections from pig herds.
Immunization trials were performed to protect pigs against F18+ E. coli. However, no protective immune response could be induced although a lot of different immunization strategies were performed. This is in contrast with F4+ E. coli infections, where oral immunization with F4 fimbriae could induce a protective mucosal immune response.
Immunization of pigs with weak virulent F18+ E. coli strains cannot be taken into practice because these strains still produce enterotoxins. Oral administration of F18 fimbriae, encapsulated in poly(D,L-lactide-co-glycolide) (PLGA) particles cannot induce significant amounts of F18-specific serum antibodies nor a reduced colonization upon F18+ E. coli challenge. In a more recent study, only a low F18-specific immune response could be induced after oral and intranasal immunization with high concentrations of purified F18 fimbriae (30 mg and 1 mg, respectively) in the presence of the adjuvant LT(R192G) or CTA1-DD, respectively (Verdonck F, Cox E, van Gog K, Van der Stede Y, Duchateau L, Deprez P, Goddeeris B M, 2002. Vaccine 20:2995-3004). In another study delivery of the F18 fimbrial adhesin FedF to the intestinal mucosa was improved by covalent coupling of MBPFedF to F4 fimbriae (Tiels P., Verdonck F., Coddens A., Goddeeris B., Cox E., 2008. Vaccine, 26, 2154-2163). The coupled product could induce a systemic and local FedF-specific immune response and led to a reduction in excretion upon challenge with F18+ E. coli. However, no complete protection was obtained.
Also various methods for passive protection against F18+ E. coli infections in pigs were tested in the past.
F18-specific antibodies present in chicken egg yolk after immunization of chickens with F18 fimbriae were administered to pigs. They could reduce the shedding of F18+ E. coli and diminished the amount of diarrhea and death after experimental infection of pigs. Verotoxin II-specific antibodies injected intramuscularly one day before weaning could protect pigs from symptoms of edema disease. However, since no reduction in colonization of F18+ E. coli is obtained, F18+ E. coli strains will still emerge in the environment. Therefore, it is better to interfere with the initial attachment of the bacterium with the host, which is the first step in the infection process.
Colonization of the porcine gut with F18+ E. coli could be reduced by oral administration of non-immune plasma powder, derived from blood of healthy slaughter pigs. This protective effect of porcine plasma powder was suggested to be due to glycan moieties present on plasma glycoproteins, although this needs further proof Another approach against F18+ E. coli could be replacing some or all of the plant-based proteins supplemented in pig feed by animal-based proteins (U.S. Pat. No. 6,355,859 published on 12 Apr. 2002: Interactions between genotype and diet in swine that prevent E. coli associated intestinal disease). Suitable animal-based proteins include milk, blood plasma and fish meal. A drawback of these last methods is that the concentration of the carbohydrates required for effective inhibition of adhesion in vitro or in vivo is high.
Although the biological function of histo-blood group antigens is not clarified yet, it has been reported before that histo-blood group antigens can act as receptors for pathogenic organisms. Several bacteria, including Helicobacter pylori and Campylobacter jejuni, have been shown to utilise blood group determinants as receptors to facilitate their colonisation. While C. jejuni binds to the blood group H type 2 determinant (Ruiz-Palacios G M, Cervantes L E, Ramos P, Chavez-Munguia B, Newburg D S, 2003. J Biol Chem. 278(16):14112-20), the primary receptor for H. pylori is the Leb determinant (Fucα2Galβ3(Fucα4)GlcNAc), along with a lower affinity binding to the H type 1 determinant (Borén T, Falk P, Roth K A, Larson G, Normark S, 1993. Science. 262(5141):1892-5). Binding to Leb and the H type 1 determinant is mediated by the BabA adhesion. Recently the adaption of the BabA adhesin to the fucosylated blood group antigens most prevalent in the local population was described. In Europe and the US, where the blood group ABO phenotypes are common in the population, the H. pylori strains (designated generalist strains) bind to blood group A, B, and O type 1 determinants However, in populations such as the South American native population, which only have the blood group O phenotype, the H. pylori strains (designated specialist strains) bind only to the blood group O determinants (Leb and the H type 1).
Viral binding to blood group antigens has also been reported. H type 2 histo-blood group trisaccharides on rabbit epithelium are recognized by Rabbit Hemorrhagic Disease Virus (RHDV) and H type 1 and H types 3/4 on gastroduodenal epithelial cells of secretor individuals are used by Norwalk virus as ligands.
The synthesis of histo-blood group antigens requires several glycosyltransferases acting on precursor oligosaccharides, such as type 1 (Galβ3GlcNAcβ-R) and type 2 precursor chains (Galβ4GlcNAcβ-R). These precursors are converted into H antigens by the action of an α(1,2)fucosyltransferase adding a fucose in an α1,2 linkage. In pigs, two genes encoding fucosyltransferases, FUT1 and FUT2, have been identified, but only the polymorphism on base pair 307 of FUT1 was found to be associated with resistance to F18+ E. coli infections. Human and porcine FUT1 preferentially fucosylate type 2 precursor chains in vitro pointing to a link between type 2 carbohydrate chains and the F18R. Nevertheless, some studies indicate that FUT1 can be responsible for α1,2 fucosylation of type 1 chains in vitro and in vivo (Liu Y H, Fujitani N, Koda Y, Kimura H, 1998. J Histochem Cytochem. 46:69-76, Mathieu S, Prorok M, Benoliel A M, Uch R, Langlet C, Bongrand P, Gerolami R, El-Battari A, 2004. Am J Pathol. 164:371-383).
A previous study of Snoeck et al. (Veterinary Microbiology 100, (2004) 241-246) describes the inhibition of adhesion of F18+ E. coli to piglet intestinal villous enterocytes by monoclonal antibody against blood group H-2 antigens. A publication of Coddens et al. (Veterinary Microbiology 122, (2007) 332-341) reports that the age dependent expression of the F18+ E. coli receptor on porcine gut epithelial cells is positively correlated with the presence of H histo-blood group antigens on type 2 core chains.
These previous studies are correlative and suggest that the F18 receptor contains the blood group antigen H-2 in its carbohydrate structure. However, the identity and nature of the F18R molecule remained unclear.
Unexpectedly, the present invention demonstrate that the FedF adhesin of F18 fimbriated E. coli recognizes glycosphingolipids (GSLs) with blood group A, B or H determinants on type 1 and type 4 core chains while blood group A, B or H determinants on type 2 core chains are not recognized. Pure A type 3 GSL's were not available for testing.
Thus the problem solved by the present invention is the identification of carbohydrate structures that are recognized by F18+ E. coli. Identification of the interaction of the above-mentioned A/B/H blood group determinants on type 1 and type 4 core chains with F18+ E. coli can be applied for receptor-based prevention strategies against F18+ E. coli infections in pigs. In contrast to antibiotics, administration of specific inhibitors of the interaction between F18+ E. coli and the F18 receptor is a gentle and safe way to counteract bacterial infections. Furthermore, since receptor-based prevention strategies do not act by killing or arresting the growth of the infectious agent, it is assumed that strains resistant to such agents will arise to a lesser extent compared to strains resistant to antibiotics. Identification of the carbohydrate structures that are recognized by F18+ E. coli makes it possible to supplement pig feed with very specific molecules that interfere with the interaction between F18+ E. coli and the gut epithelium at low concentrations when compared with less specific inhibitors such as e.g. blood plasma, or the combination therapies such as for example described in WO2004/002495.